Ion mobility analysis of lipoproteins

ABSTRACT

A medical diagnostic method and instrumentation system for analyzing noncovalently bonded agglomerated biological particles is described. The method and system comprises: a method of preparation for the biological particles; an electrospray generator; an alpha particle radiation source; a differential mobility analyzer; a particle counter; and data acquisition and analysis means. The medical device is useful for the assessment of human diseases, such as cardiac disease risk and hyperlipidemia, by rapid quantitative analysis of lipoprotein fraction densities. Initially, purification procedures are described to reduce an initial blood sample to an analytical input to the instrument. The measured sizes from the analytical sample are correlated with densities, resulting in a spectrum of lipoprotein densities. The lipoprotein density distribution can then be used to characterize cardiac and other lipid-related health risks.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/293,610, now U.S. Pat. No. 7,259,018, filed Nov. 12, 2002, entitled“Ion Mobility Analysis of Biological Particles,” which claims priorityto U.S. provisional patent application No. 60/338,214, filed Nov. 13,2001, entitled “Ion Mobility Analysis of Biological Particles,” each ofthese references are hereby incorporated by reference.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with U.S. Government support under ContractNumber DE-AC03-765F00098 between the U.S. Department of Energy and TheRegents of the University of California for the management and operationof the Lawrence Berkeley National Laboratory. The U.S. Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to particle size analysis and,further, to analysis of biological particles for diagnostic purposesutilizing traditional particulate size or mobility measurement devices.One aspect of the present invention more particularly relates to medicaldiagnostics for the quantitative and qualitative analysis of lipoproteinclasses and subclasses and their relationship to the assignment ofcoronary heart disease and other lipid-related health risks.

2. Description of the Relevant Art

Introduction

In clinical practice, lipoprotein particle measurements are used toassess cardiovascular and other lipid-related health risks, determinetreatment protocols and track the efficacy of treatment regimens.Lipoprotein particles comprise macromolecules that package cholesteroland other biochemicals, enabling them to be transported through theblood stream. The size distribution of lipoprotein particles variesamong individuals due to both genetic and nongenetic influences. Thediameters of lipoprotein particles typically range from about 7 nm toabout 120 nm. In this diameter size range, there exist subfractions ofthe particles that are important predictors of cardiovascular disease.For instance, very low density lipoproteins transport triglycerides inthe blood stream; high very low density lipoprotein levels in the bloodstream are indicative of hypertriglyceremia. These subfractions areidentified by analytical techniques that display the quantity ofmaterial as a function of lipoprotein particle size or density.

Standard Plasma Lipid and Lipoprotein Cholesterol Measurement Techniques

Typical standard lipid measurements include fasting total cholesterol,triglyceride, as well as HDL and LDL cholesterol. Currently, the mostwidely used method for measuring LDL cholesterol is the indirectFriedewald method (Friedewald, et al., Clin. Chem. Vol. 18, pp. 499-502,1972). The Friedewald assay method requires three steps: 1)determination of plasma triglyceride (TG) and total cholesterol (TC), 2)precipitation of VLDL and LDL, and 3) quantitation of HDL cholesterol(HDLC). Using an estimate for VLDLC as one-fifth of plasma triglycerides

$( \frac{T\; G}{5} ),$

the LDL cholesterol concentration (LDLC) is calculated by the formula:LDLC=TC−(HDLC+VLDLC). While generally useful, the Friedewald method islimited in its accuracy in specific cases. Errors can occur in any ofthe three steps, in part because this method requires that differentprocedures be used in each step. The Friedewald method is to a degreeindirect, as it presumes that VLDLC concentration is one-fifth that ofplasma triglycerides. When the VLDL of some patients deviates from thisratio, further inaccuracies occur. Ultracentrifugation must be employedfor separation and subsequent determination of LDL cholesterol for somesamples, since the Friedewald method cannot be used for patients with TGover 400 mg/dL.

Procedures for Lipoprotein Subspecies Analysis

Presently, the predominant methods for lipoprotein subspecies analysisinclude nuclear magnetic resonance, the vertical auto profile, andElectrophoretic gel separation. Each of these methods will be brieflydiscussed below.

Nuclear Magnetic Resonance

Otvos teaches a nuclear magnetic resonance (NMR) procedure fordetermining the concentrations of lipoprotein subclasses, which hasgreater accuracy than Friedewald (U.S. Pat. No. 5,343,389, issued Aug.30, 1994). Otvos initially obtains the NMR chemical shift spectrum of ablood plasma or serum sample. The observed spectrum of the entire plasmasample is then matched with the known weighted sums of NMR spectra oflipoprotein subclasses, which are stored in a computer software program.The weight factors that give the best fit between the sample spectrumand the calculated spectrum are then used to estimate the concentrationsof constituent lipoprotein subclasses in the blood sample. Thisprocedure has the additional advantage of being rapid.

Vertical Auto Profile

Another lipoprotein subfraction determination method that is usedclinically is the Vertical Auto Profile (VAP), (Kulkarni, et al., J.Lip. Res., Vol. 35, pp. 159-168, 1994). In the Vertical Auto Profilemethod, a flow analyzer is used for the enzymatic analysis ofcholesterol in lipoprotein classes separated by a short spin singlevertical ultracentrifugation, with subsequent spectrophotometry andsoftware analysis of the resulting data. While a useful advance, thistechnique does not resolve the LDL into all seven subspecies identifiedby electrophoretic gradient gel separation.

Electrophoretic Gradient Gel Separation

The gel separation method is demonstrated in U.S. Pat. No. 5,925,229,issued Jul. 20, 1999, by R. M. Krauss, et al., entitled “Low DensityLipoprotein Fraction Assay for Cardiac Disease Risk,” (the '229 patent),which is hereby incorporated by reference. In the '229 patent, agradient gel electrophoresis procedure for the separation of LDLsubclasses is disclosed. The LDL fractions are separated by gradient gelelectrophoresis, producing results that are comparable to those obtainedby ultracentrifuge. This method generates a fine resolution of LDLsubclasses, and is used principally by research laboratories. However,gradient gel electrophoresis can take many hours to complete. It wouldbe useful if gradient gel electrophoresis separation times could beshortened and the analysis simplified so that high resolution lipidanalysis could be used in clinical laboratories as part of a routinescreening of blood samples, and to assign a risk factor for coronaryartery disease.

The gel separation method, which depends on uniform staining of allcomponents that are subsequently optically measured, suffers fromnonuniform chromogenicity. That is, not all lipoprotein particles stainequally well. The differential stain uptake produces erroneousquantitative results, in that a less staining peak may be read at alower value than is actually present. Additionally, the nonuniformchromogenicity can result in erroneous qualitative results, in thatmeasured peaks may be skewed to a sufficient degree as to confuse oneclass or subclass of lipoprotein with another.

A high-resolution assay for measuring all subclasses of LDL as well asVLDL, IDL, HDL, and chylomicron particles that would be accurate,direct, and complete, would be an important innovation in lipidmeasurement technology. If inexpensive and convenient, such an assaycould be employed not only in research laboratories, but also in aclinical laboratory setting. Ideally, clinicians could use thisinformation to improve current estimation of coronary disease risk andmake appropriate medical risk management decisions based on the assay.

SUMMARY OF THE INVENTION

This invention provides: 1) a method for preparing plasma samples fordifferential mobility analysis (“DMA”), 2) a technique for measuring thesize or density distribution of biological particles (preferablylipoprotein particles) based on the measured particle mobility countingrate for the number of biological particles counted per second at aspecific selected mobility as a function of size,

${\frac{n^{+}}{t}}_{s},$

or density,

${\frac{n^{+}}{t}}_{\rho},$

of ionized biological particles sprayed into a fluid (such as air), 3) aDMA device for measuring the number of biological particles in each sizeclass and subclass, 4) an algorithm for predicting the risk of coronaryheart disease (CHD) using the biological particle (preferablylipoprotein) size or density distribution information compared toreference data for low risk, intermediate risk, and high risk patients,and 5) a particular method for determining whether a sample correspondsto a Type A, AB, or B pattern, which respectively has an associated lowrisk, an intermediate risk, and a high risk for CHD. Blood plasmasamples are processed to separate all classes and subclasses oflipoprotein particles from bulk blood serum using steps designedultimately for transferring the particles into the gas phase forsubsequent mobility analysis. The technique preserves lipoproteinparticle composition and size, and introduces no contamination into thelipoprotein particles. The electrospray process initially produceselectrospray droplets with many different charge states. Thesemultiply-charged droplets are passed in close proximity to an alpharadiation source, which produces alpha particles, or free secondaryelectrons, that reduces the charge state of the electrospray droplets toeither neutral or single positive charges. The charged droplets mayalternatively be treated in other ways to achieve a uniform charge stateof no more than a single positive charge.

Subsequent volatilization of the droplet diluent results in individuallipoprotein particles having either a neutral or a single positivecharge. By scanning a high voltage selection voltage that modifies amobility selection, lipoprotein particle gas-phase mobility can bemeasured over a range of mobilities. When the scanned mobilities arecounted over a scan range, a mobility distribution can be developed.Personnel skilled in reading the lipoprotein mobility distribution canmake direct conclusions regarding the cardiac risk for the patient.

An inverse linear relationship between lipoprotein mobility and sizeallows the mobility distribution data to be converted to a particle sizedistribution, thereby providing a way to display a spectrum, ordistribution, of the number of particles counted per unit time vs.particle size. Given a constant time basis, this measurement can beregarded as number of particles vs. particle size, and can be relatedback to a quantitative fractional distribution of lipoproteins in theoriginal sample. The resulting lipoprotein particle size distribution iscomprised of component regions. These regions, in turn, distinguish thequantity and types of lipoprotein particles in the original serumsample. By determining the relative location of certain peaks in certainof the regions, a prediction can be made as to the cardiac risk. Thesize distribution may additionally be converted into a distribution ofthe number of particles vs. density for more traditional comparison oflipoprotein densities.

The present invention provides a method for determining a cardiovasculardisease in a patient comprising providing a biological sample from apatient; obtaining a mobility distribution of charged lipoproteinparticles from the sample by electrospraying the lipoprotein particlesso that the particles are provided in a charged state; delivering thelipoprotein particles to a differential mobility analyzer; comparing themobility distribution of lipoprotein particles from the patient to areference lipoprotein particle mobility distribution; and determiningwhether the patient has a cardiovascular disease based on thecomparison. In one embodiment, the charged lipoprotein particles are atleast one of VLDL, IDL, LDL, HDL or their subclasses. In anotherembodiment, the biological sample is blood, plasma or serum. In anotherembodiment, the charged lipoprotein particles are classified on thebasis of the mobility distribution. The method may further compriseconverting the mobility distribution of charge reduced lipoproteins toat least one of the following measurements prior to determining thecardiovascular disease of the patient: a particle mobility, a particlesize, a particle density, a particle mass, a number of particles in asize interval and an amount of particle mass in a size interval. In oneembodiment, the cardiovascular disease is coronary heart disease. Inanother embodiment, step (d) is carried out by a deterministicalgorithm.

The present invention also provides a method for determining acardiovascular condition in a patient, comprising providing a biologicalsample from a patient; obtaining a mobility distribution of chargedlipoprotein particles from the sample by electrospraying the lipoproteinparticles so that the particles are provided in a charged state;delivering the lipoprotein particles to a differential mobilityanalyzer; comparing the mobility distribution of lipoprotein particlesfrom the patient to a reference lipoprotein particle mobilitydistribution; and determining whether the patient has a cardiovascularcondition based on the comparison. In one embodiment, the chargedlipoprotein particles are at least one of VLDL, IDL, LDL, HDL or theirsubclasses. In another embodiment, the biological sample is blood,plasma or serum. In another embodiment, the charged lipoproteinparticles are classified on the basis of the mobility distribution. Themethod may further comprise converting the mobility distribution ofcharge reduced lipoproteins to at least one of the followingmeasurements prior to determining the cardiovascular condition of thepatient: a particle mobility, a particle size, a particle density, aparticle mass, a number of particles in a size interval and an amount ofparticle mass in a size interval. In another embodiment, thecardiovascular condition is cardiac disease or a predisposition todevelop cardiovascular disease.

The present invention also provides a method for determining apredisposition to developing a cardiovascular disease or condition in apatient comprising providing a biological sample from a patient;obtaining a mobility distribution of charged lipoprotein particles fromthe sample by electrospraying the lipoprotein particles so that theparticles are provided in a charged state; delivering the lipoproteinparticles to a differential mobility analyzer; comparing the mobilitydistribution of lipoprotein particles from the patient to a referencelipoprotein particle mobility distribution; and determining whether thepatient has a predisposition to develop a cardiovascular disease orcondition based on the comparison. In one embodiment, the chargedlipoprotein particles are at least one of VLDL, IDL, LDL, HDL or theirsubclasses. In another embodiment, the sample is blood, plasma or serum.In another embodiment, the charged lipoprotein particles are classifiedon the basis of the mobility distribution. The method may furthercomprise converting the mobility distribution of charge reducedlipoproteins to at least one of the following measurements prior todetermining the predisposition to develop the cardiovascular disease orcondition of the patient: a particle mobility, a particle size, aparticle density, a particle mass, a number of particles in a sizeinterval and an amount of particle mass in a size interval. In anotherembodiment, step (d) is carried out by a deterministic algorithm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an overview of the ion mobility analysis of biologicalparticles hardware and analytical sequence.

FIG. 2 is a table of major lipoprotein classes, subclasses, densitiesand particle sizes as reported from gel electrophoresis results.

FIG. 3 is a close up cross-sectional view of a properly operatingaxisymmetric electrospray capillary with a stable Taylor cone.

FIG. 4 is a cross sectional view through the centerline of anaxisymmetric differential mobility analyzer.

FIG. 5 is a scanning differential mobility analyzer output ofmass-offset lipoprotein size distributions obtained from sixindividuals.

FIG. 6 is a graph indicating the approximately inverse linearrelationship between lipoprotein particle diameter, and the estimatedlipoprotein sample density.

FIG. 7 is a graph indicating the approximately linear relationshipbetween lipoprotein particle count, and the corresponding lipoproteinsample particle count.

FIG. 8 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.000 g/cm³,corresponding to VLDL I.

FIG. 9 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.003 g/cm³,corresponding to VLDL I.

FIG. 10 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.005 g/cm³,corresponding to VLDL I.

FIG. 11 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0075 g/cm³,corresponding to VLDL II.

FIG. 12 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0105 g/cm³,corresponding to IDL I.

FIG. 13 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0161 g/cm³,corresponding to IDL II.

FIG. 14 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0217 g/cm³,corresponding to LDL I.

FIG. 15 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0273 g/cm³,corresponding to LDL Ha.

FIG. 16 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0330 g/cm³,corresponding to LDL IIb.

FIG. 17 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0386 g/cm³,corresponding to LDL

FIG. 18 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0442 g/cm³,corresponding to LDL IVa.

FIG. 19 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0498 g/cm³,corresponding to LDL IVa.

FIG. 20 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0692 g/cm³,corresponding to HDL IIb.

FIG. 21 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0767 g/cm³,corresponding to HDL IIb.

FIG. 22 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0844 g/cm³,corresponding to HDL IIb.

FIG. 23 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.090 g/cm³,corresponding to HDL IIb.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

“Biological particle” means a material having a non-covalently boundassembly of molecules derived from a living source. Examples arelipoprotein particles assembled from apolipoproteins and lipids; viralcomponents assembled from non-covalently bound coat proteins andglycoproteins; immune complexes assembled from antibodies and theircognate antigens, etc., but not entire cells.

“Physiological sample” means a sample obtained from an organism, such asblood, tissue, pulp, cytoplasm, etc.

“CHD” means coronary heart disease.

“VLDL, IDL, LDL, and HDL” are described by class name, acronym,subclass, and density range as shown in FIG. 2.

“Chylomicrons” means biological particles of size 70-120 nm, withcorresponding densities of less than 1.006 g/mL.

“Differential Mobility Analyzer” means a device for classifying chargedparticles on the basis of their ion electrical mobility. When theparticles have a known uniform charge, the size of the particlesclassified may be determined from their mobility.

“Lp(a)” means biological particles consisting of LDL covalently attachedto the protein lipoprotein A.

“Lipoprotein particles” means particles obtained from mammalian blood,comprising apolipoproteins biologically assembled with noncovalent bondsto package cholesterol and lipids. Lipoprotein particles preferablyrefer to biological particles having a size range of 7 to 1200 nm.Lipoprotein particles, as used herein, essentially include VLDL, IDL,LDL, HDL and chylomicrons.

“Centrifugation” means separation or analysis of substances in asolution as a function of density and density-related molecular weightby subjecting the solution to a centrifugal force generated byhigh-speed rotation in an appropriate instrument.

“Pattern A” is the designation applied to individuals having acharacteristic lipoprotein particle distribution indicating a relativelylow risk for CHD, as illustrated in FIG. 5.

“Pattern B” is the designation applied to individuals having acharacteristic lipoprotein particle distribution indicating a relativelyhigh risk for CHD, as illustrated in FIG. 5.

“Pattern AB” is the designation applied to individuals having acharacteristic lipoprotein particle distribution indicating anintermediate risk for CHD, as illustrated in FIG. 5. Pattern AB has adistribution with some of the characteristics of both Pattern A andPattern B.

“Predisposition” as used herein is substantially synonymous with risk,inclination, tendency, predilection, or susceptibility.

“Reference Size Distribution” means a lipoprotein particle sizedistribution such as Patterns A, B, or AB as further discussed inconnection with FIG. 5.

“Computer readable medium” means any source of organized informationthat may be processed by a computer, including but not limited to: amagnetically readable storage system; optically readable storage mediasuch as punch cards or printed matter readable by direct methods,optical reflectance and transmission scanning, or methods of opticalcharacter recognition; other optical storage media such as a compactdisc (CD), digital versatile disc (DVD), rewritable CD and/or DVD;electrically readable media such as programmable read only memories(PROMs), electrically erasable programmable read only memories(EEPROMs), field programmable gate arrays (FPGAs), flash random accessmemory (flash RAM); and remotely transmitted information transmitted byelectromagnetic or optical methods.

“Distribution” means a generalized function of one or more variables,and commonly depicted as a scatter chart, graph, plot, or histogram.

Overview of Ion Mobility Analysis of Biological Particles

FIG. 1 depicts the major functional components involved in biologicalparticle ion mobility analysis. A sample is placed in a microcentrifugetube 410, which is then placed in an centrifuge 420 to removeundesirable components, such as Lp(a), and other cellular components asdescribed below in Section D “Preparation of Purified LipoproteinParticle Samples.” After removal of cellular components and Lp(a), aswell as further density ultracentrifugation to select only lipoproteinmaterials, a sample solution 455 is placed in a testing microcentrifugetube 450, which is in turn placed in a pressurized chamber 460. A highvoltage variable power supply 430, positively biases the pressurizedchamber 460, and the sample solution 455, through a Pt wire 435. Thepositive bias and higher relative pressure causes a capillary 100 toform a Taylor cone 150 emitting particle droplets from the pressurizedchamber 460. FIG. 3 more fully details the capillary 100.

Still referring to FIG. 1, once the droplets are formed, a dry gas 210propels the droplets into an emission region of an alpha radiationsource 480, which reduces the charge state of the droplets to no morethan one positive charge per droplet as the droplets containingcomponents other than the volatile diluent dry to single particles. Thecharged droplets may alternatively be treated with other methods toachieve a uniform charge state of no more than a single positive charge.One such other method is to use an alternating current corona, whichproduces secondary electrons having the same charge state reduction asan alpha source.

After charge reduction, the dry gas 210 propels the particles into adifferential mobility analyzer 200, which is shown in more detail inFIG. 4. A laminar flow excess gas 220, as well as an additional dry gas230 flow is introduced to match the velocity of the dry gas 210 flow. Byvarying a high voltage power supply 205, the particles carried by thecombined flows are selected into a mobility selected particle 240 flow,which in turn flows into a particle counter 490.

Particle counter 490 is preferably in communication with a computersystem (not shown) having storage capability onto one or more computerreadable media for recording a scanned distribution output of thedifferently sized particles. As discussed below under Section F“Differential Mobility Analysis,” the single positively chargedparticles, the fixed dry gas 210 and additional dry gas 230 flows, andparticular voltage setting of the scanned high voltage power supply 205,classifies a selected particle stream 285 to enter into an exitingannular selection slit 290, the selected particle stream 285 havingparticles with a particular mobility and corresponding particle size.

One feature of this invention is that loose, non-covalently bondedbiological particles can be processed through this system without losingtheir biological identity or breaking apart. In particular, lipoproteinparticles (e.g. VLDL, IDL, LDL, and HDL and their subclasses) can beprocessed in such a fashion so that their particle mobilities, and hencesize and density, can be quickly determined, without lipoproteinparticle breakdown.

Once a relationship is known between particle size and density, size ormobility distributions may be converted into distributions of: aparticle mass, a density μg/cm³ of original plasma, a number ofparticles in a size interval, and an amount of particle mass in a sizeinterval.

Lipoprotein Particles

Lipoproteins are complex macromolecules found in human blood plasma thatpackage lipids into lipoprotein particles. Among other functions,lipoprotein particles in the circulatory system transport cholesterolfor cellular use. Lipoprotein particles are subdivided into a variety ofclasses and subclasses based on density as well as particle size.Lipoprotein particle density can be determined directly by equilibriumdensity ultracentrifugation and analytic ultracentrifugation.Lipoprotein particle density may also be determined indirectly based onparticle size and a known relationship between particle size anddensity. Particle size may be determined by several methods includinggel electrophoresis.

FIG. 2 is a table of the present standard classes and subclasses thathave been assigned to various lipoprotein fractions using traditionalgel electrophoresis measurements: very low density lipoproteins (VLDLs)with subclasses VLDL I and II, intermediate density lipoproteins (IDLs)with subclasses IDL I and II, low density lipoproteins (LDLs) and highdensity lipoproteins (HDLs), which typically includes severalsubclasses, such as HDL IIa, IIb, IIIc, IIIb, and IIIc.

Chylomicrons are not included in FIG. 2, as chylomicrons have not beenfound to have any clinical significance in the prediction of heartdisease.

Ultracentrifugally isolated lipoprotein particles can be analyzed forflotation properties by analytic ultracentrifugation in two differentsalt density backgrounds, allowing for the determination of hydrated LDLparticle density, as shown in Lindgren, et. al, Blood L is ids andLipoproteins: Quantitation, Composition and Metabolism, Ed. G. L.Nelson, Wiley, 1992, p. 181-274, which is incorporated herein byreference.

The LDL class, as indicated in FIG. 2, can be further divided into sevensubclasses based on density or particle diameter by using a preparativeseparation technique known as equilibrium density gradientultracentrifugation (EDGU). It is known that elevated levels of specificLDL subclasses, LDL-IIIa, IIIb, IVa and IVb, is a clinical finding whichcorrelates closely with increased risk for CHD, includingatherosclerosis.

Determination of the total serum cholesterol level and the levels ofcholesterol in the LDL and HDL fractions are routinely used asdiagnostic tests for coronary heart disease risk. Lipoprotein class andsubclass distribution is a more predictive test, however, since it isexpensive and time-consuming, it is typically ordered by physicians onlyfor a limited number of patients.

It should be noted that the values used in FIG. 2 for sizes aredetermined by gel electrophoresis methods. With the ion mobility methodsdisclosed here, it has been observed that all measurements oflipoprotein particle diameter obtained with ion mobility are shifted tosmaller diameters compared to the data obtained with gelelectrophoresis. It is surmised that this difference is caused bycalibration of the gels. The shift appears to be linearly related withapproximately:

0.86*gel diameter=ion mobility diameter

The observed differences between ion mobility diameters and gelelectrophoresis diameters may also be due to the fact that thelipoprotein particles are distorted as they bump into the gel matrixunder the influence of the electrophoresis gel's intrinsic impressedelectric field. The size difference may also be due to historical dataused to convert particle density (obtained from analytic ultracentrifugeseparations) to particle size obtained from electron microscopy. FIG. 2presents the lipoprotein sizes accepted for gel electrophoresis runs aspublished in common scientific literature. It is the inventors' opinionthat ion mobility sizes may be more accurate than gel sizes, however,this needs to be confirmed.

Preparation of Purified Lipoprotein Particle Samples

Generally, sample preparation comprises the following steps: taking ablood sample, separation of the blood plasma from the cellular matter,removal of non-lipoprotein proteins from the plasma by one of severalmethods, such as ultracentrifugation or adsorption on affinity gels,dialysis filtration to remove density adjusting components that wouldaffect particle volatility and/or size, addition of volatile reagents tofacilitate electrospray droplet formation, dilution of lipoproteinparticle sample solution, and adjustment of sample solution pH.

A 50 to 100 μL fasting blood sample is initially taken. Chylomicrons arenot typically present in people who have been fasting for a period of atleast 12 hours, so overlap of VLDL particle sizes and chylomicron sizesis eliminated by fasting. The sample is then initially spun in acentrifuge. The sample is preferably spun for approximately 10 minutesat 2000 gravities, sufficient to remove the cellular components from thesample. During this process, the more dense cellular components stratifyat the bottom of the sample. A remaining less dense plasma specimen ontop is then drawn off.

The plasma specimen is then density-adjusted to a specific density usinghigh purity solutions of sodium chloride (NaCl) and sodium bromide(NaBr). In one embodiment, the specific density is chosen to be greaterthan or equal to the highest density of the lipoprotein material to beanalyzed, so that the lipoprotein material floats when densitystratified. These densities may be found in the FIG. 2 table oflipoprotein classes, subclasses, densities, and sizes. By sequentiallycentrifuging from lowest density to highest density of the densityadjustment, the various classes and subclasses of lipoproteins may besequentially extracted.

In the preferred method used in this invention, instead of a series ofsequential density adjustments followed by centrifugation, just a singledensity adjustment using NaCl and NaBr salts is performed. A singlespecific density is chosen for density adjustment in the preferredembodiment. The sample density adjustment can be selected within therange of 1-1.21 g/mL according to the densities in FIG. 2 to separate aclass of lipoproteins having equal or lesser density. In the preferredembodiment, a density adjustment to 1.21 g/mL is made. In this manner,all HDLs, IDLs, LDLs, and VLDLs are simultaneously extracted, since theyhave densities less than 1.21 g/mL.

The density-adjusted plasma sample is then ultracentrifuged in a commoncommercially available 100 μL ultracentrifuge tube for approximately 18hours at 100,000 gravities, or 10⁶ m/s² to separate the non-lipoproteinproteins from the lipoprotein particles. Non-lipoprotein proteins,particularly albumin, are then removed from the plasma specimen,preferably by this ultracentrifugation step. The lipoprotein particlesfloat to the top of the sample during ultracentrifugation.

Lp(a) is another type of lipoprotein particle found in serum having amolecular composition distinct from IDL and LDL particles. Lp(a) has aparticle size that overlaps with LDL and IDL particles and thereforewill interfere with particle size analysis when Lp(a) particles arepresent in serum. Although some patients have naturally occurring lowLp(a) concentrations, it is a good practice to remove the Lp(a) prior toLDL particle size measurements to preclude otherwise inaccuratemeasurements for those patients who do have significant Lp(a)concentrations. In this manner, any potential Lp(a) size interferenceproblem is avoided.

In sample solutions where Lp(a) has not been removed, up to half of theLDL measurement can be comprised of Lp(a). Therefore, beforeelectrospray analysis Lp(a) is preferably removed from the plasmasample. This removal can be achieved by solid phase absorption of theLp(a) protein using either lectin or specific antibodies attached to amatrix. Lp(a) concentration can then be measured separately (e.g. byimmunoassay) or by the difference between the immunoassay results usingabsorbed and unabsorbed plasma. Lp(a) is most preferably removed bylectin affinity chromatography. The Lp(a) removal step preferablyfollows density adjustment and ultracentrifugation. It precedes dialysisof the selected 1.21 g/mL fraction.

The lowest density, top portion, of the ultracentrifuged sample is thendrawn off for subsequent dialysis as a liquid sample containinglipoprotein particles. This liquid is then placed behind a 10,000 Daltonmolecular weight cutoff filter, to retain the lipoprotein particlesbehind the filter, yet freely allow diffusion of H₂O, NaCl and NaBrionic components. The filter, with the liquid containing the densityadjusting NaCl and NaBr, is immersed in 0.22 μm particle-filtered, 18Meg-ohm deionized water to allow the NaCl and NaBr to reachconcentration equilibrium with the surrounding water. In this fashion,the NaCl and NaBr, which otherwise would have affected particle weightduring mobility measurement, are removed, or dialyzed. In thelaboratory, it has been found sufficient to place the cutoff membrane incontact with water, and simply deposit a drop of sample to the top ofthe membrane. The NaCl and NaBr levels in the drop of sample then reachequilibrium with the water diluent.

The sample solution is then prepared in a volatile aqueous buffer atsufficiently near neutral pH (pH 7.0), so as to prevent the lipoproteinparticles from disintegrating into one or more of their constituentmolecules. The preferred buffer solution is 20-30 mM, preferably 25 mM,ammonium acetate in particle-filtered 18 Meg-ohm deionized water with pHadjusted to neutral with high purity ammonium hydroxide. It is criticalto prepare the ammonium acetate and ammonium hydroxide solutions withvery high purity reagents in order to prevent contamination of thelipoprotein particles, which would subsequently affect the particlemobility measurements.

The volatile aqueous buffer serves three principal functions: 1) todilute the sample sufficiently so that, when electrosprayed, only onelipoprotein particle is contained within each electrospray droplet, 2)to provide for electrical conductivity so that the capillary bore samplecontents are uniformly at approximate equipotential with the highvoltage power source, and 3) to impart a higher vapor pressure to thebulk solution, thereby improving volatility of the diluted sample andhastening droplet drying time. Although ammonium acetate has been usedhere, people skilled in the art could arrive at several alternativeformulations achieving the same three functions.

The amount of insoluble material and the residue remaining afterdilution and subsequent volatilization are important specifications forchoosing a particular quality of reagent. These quantities should beminimized; preferably each reagent should have less than 0.005 weightpercent of impurities. Subsequently, the lipoprotein sample is eitherprepared in solution with the reagent or diluted with this reagent toabout 10¹¹ particles per mL or less.

At lipoprotein particle concentrations of less than 10¹¹ particles permL, no more than one lipoprotein particle should be present in a singleelectrospray droplet. By designing the dilution process to have only onelipoprotein particle in an electrospray droplet, a potential aliasingproblem of artificially combining lipoprotein particle clusters, andthereby detecting the lumped cluster as a larger lipoprotein, isavoided. Restating this issue, if two small weight lipoprotein particlesarrive in a single droplet, then the resulting size measurement of thecompound particle is much larger, and is not representative of theoriginal lipoprotein sample. Thus, if aliasing were to occur, two ormore small HDL particles could be measured as a single VLDL particle,distorting the mobility measurement, and further indicating an incorrectcorresponding particle size distribution.

The lipoprotein particles suspended in the ammonium acetate solution arethen electrosprayed. The electrospray is fed by pumping the samplesolution at a rate of about 50 mL per minute through a small capillarytube, preferably an Osage number 062442 capillary. Between 20 and 50 μLof sample solution is placed in a microcentrifuge tube and the entranceto a silica capillary having a 20 μm interior diameter is placed intothis sample solution. The microcentrifuge sample tube and the entranceend to the capillary are sealed in a positive pressure container. Apositive differential pressure, on the order of 3 psig, is appliedacross the capillary, producing a flow of sample solution through thecapillary. The outlet side of the capillary, maintained at approximatelyambient atmospheric pressure, is inserted into an electrospray dropletgenerator, preferably a TSI Model 3480 Electrospray Aerosol Generator(TSI, Incorporated, St. Paul, Minn.).

Electrospray of Samples

The electrospray capillary used in this invention is shownaxisymmetrically in FIG. 3. The capillary body 100 has a hollow core,through which a sample solution 130 is transported. It is preferred touse a capillary 100 that has been passivated with a coating such as amethyl derivatization along the hollow core of the interior bore 110 ofthe capillary 100, so that substances found in the sample solution 130are not adsorbed as the sample solution 130 is electrosprayed, as suchadhesion would tend to clog the capillary, rendering it unusable untilsuccessfully cleaned. It has been found that a capillary derivatizedwith a methylating reagent replaces silanol and Si—O— groups on theinterior bore 110 with methyl groups, which thereby minimizes adsorptionof sample onto the capillary, and subsequent clogging.

Stable electrosprays are obtained when the capillary 100 is about 25 cmlong and a positive voltage of about 2 kV is applied to the sample 130.This is accomplished, as shown in FIG. 1, by placing a Pt wire 435 intothe testing microcentrifuge tube 450 and in electrical contact with thesample solution 435. The Pt wire 435 is connected to the positivepolarity of a high voltage variable power supply 430. It is alsonecessary to taper the wall of the outlet end of the capillary 120 toproduce a truncated flat-tipped cone 140 with about a 90° includedtaper. The tapered area of the capillary thus resembles a truncatedcone.

Refer now to FIG. 3. The tapered outlet end 120 of the capillary 100 isproduced by placing a capillary 100 securely into a pin-vise mounted ona 50 rpm DC motor, and subsequently grinding an approximately 45° anglefrom the longitudinal direction of the capillary.

Electrospray occurs at the flat-topped tapered outlet end of thecapillary 120. The meniscus of the exiting sample 130 liquid takes onthe characteristic shape of a Taylor cone 150, typical of stableelectrosprays, but only a sharp-tipped cone 160 can be seen during theelectrospray of highly conductive liquids typically used because thedroplet stream 170 that forms is comprised of droplets too tiny toscatter light and thus be microscopically observed.

The droplet stream 170 is carried into a small chamber by a laminar flowof CO₂ and air, established according to TSI factory recommendations,where they are exposed to an alpha radiation source 480 (as previouslyshown in FIG. 1), which, as the droplet diluent evaporates, lowers thedroplet net charge state to zero or one. The initial droplet size istypically approximately 150 nm in diameter.

As discussed in Section B “Overview of Ion Mobility Analysis ofBiological Particles,” the droplets dry rapidly in the flow of CO₂ andair and desolvate forming neutral or singly charged lipoproteinparticles. The lipoprotein particles carry the same amount of charge asdid the droplets that were initially electrosprayed. In typicalimplementations, drying and charge reduction may take place concurrentlyto prevent Coulomb forces breaking apart droplets, and here lipoproteinparticles. The resulting gas-borne particles are primarily chargeneutral or carry one positive charge. The alpha-source charge reductionprocess produces a reliable, well-characterized stream of chargedparticles. In this case, well characterized means that, although thefraction of singly charged particles depends on particle diameter, therelationship between diameter and the fraction of the particles carryinga single charge is well established. The electrospray aerosol generatordelivers neutral and singly-charge lipoprotein particles to the input ofa differential mobility analyzer 200 where the size distribution of theparticles is determined.

Other methods may be used to ensure that an entering stream of chargedparticles exits with particles having no more than a single positivecharge. One of these methods include using an alternating current coronato produce secondary electrons having the same charge state reduction asan alpha radiation source.

Differential Mobility Analysis

Ion electrical mobility analysis is a technique to determine the size ofa charged particle undergoing analysis when the charged particle isexposed to an electric field. Below follows the analytical method usedto determine the size of the charged particle.

Ion electrical mobility is a physical property of an ion and is relatedto the velocity an ion acquires when it is subjected to an electricalfield. Electrical mobility, Z, is defined as

$Z = \frac{V}{E}$

where V=terminal velocity and E=electrical field causing particlemotion. Furthermore, particle diameter can be obtained from

$Z = \frac{{neC}_{c}}{3{{\pi\eta}d}}$

where n=number of charges on the particle (in this case a singlecharge), e=1.6×10⁻¹⁹ coulombs/charge, C_(c)=particle size dependent slipcorrection factor, η=gas viscosity, and d=particle diameter. Solving ford, we obtain

$d = {\frac{{neC}_{c}}{3{\pi\eta}}\frac{E}{V}}$

Thus we obtain an explicit relationship for particle diameter as afunction of known parameters. By setting the parameters to differentvalues, different particle diameters of the charged particles may beselected as further described below. In particular, it is easiest tovary E, the electric field strength acting upon the charged particle.

A differential mobility analyzer separates the charged input lipoproteinparticles according the their diameter and charge. A preferreddifferential mobility analyzer is a TSI model 3080 ElectrostaticClassifier.

Now referring to FIG. 4, a cross section through the centerline of atypical axisymmetric differential mobility analyzer 200 is shown. Theanalyzer is designed and operated to transmit only singly chargedpositive ions of a particular size 285 through an exiting annularselection slit 290 in the instrument. For purposes of illustration thereare several particles shown that are not at the correct selection size285: neutral particles 295, and charged smaller particles 260 andcharged larger particles 280.

The differential mobility analyzer 200 is housed in a cylinder canister275 having a hollow cylindrical area 245 with a centrally placed tube270. The centrally placed tube 270 comprises an annular selection slit290 through which particles (e.g. 285 from the hollow cylindrical area245) pass when a certain electric field exists. Particles 285 enter theannular selection slit 290 as a function of the size of the particle andthe electromotive force applied to the particle. An electromotive forceresults from the application of a selection voltage applied from highvoltage source 205 to centrally placed tube 270 and cylinder canister275. Once a particle 285 enters centrally placed tube 270 throughselection slit 290, the gas flow 230 through the centrally placed tube270 carries particle-laden gas flow 240 to a particle-counting device490 (as shown in FIG. 1).

In FIG. 4, four different types of particles are shown for purposes ofillustration. The four different symbols represent singly chargedlipoprotein particles having three different sizes 260, 280, and 285,and an uncharged particle 295. When a high voltage source 205 is appliedbetween the centrally placed tube 270 and the outer cylinder 275, thepositively charged particles begin to move towards the centrally placedtube 270 at a radial velocity determined by the balance between theforces exerted by the electric field strength and the viscous resistingforce due to the drag of the particle moving through the ambient medium.

Since the viscous drag is related to the size of the particle, for thesame electromotive force, a smaller particle, having a smaller crosssectional area, will have a smaller drag, will be most affected by theelectromotive force, and will consequently have a higher mobility.Correspondingly, a larger particle will have a larger cross sectionalarea, and will be least affected by the same electromotive force, andconsequently have a lower mobility.

The axial gas velocity moving from the top of the analyzer 225 to thebottom of the analyzer 235 influences the location where the particlesimpinge the centrally placed tube 270 by vector velocity componentaddition. FIG. 4 shows that one of the types of particle ions 285 has amobility that deposits it into a detection slit 290 that leads theselected ions away to a detector 490. That is, only a small distributionof particle sizes distributed about the selected particle size exitsdetection slit 290 at a specified high voltage 205 at a specific laminargas velocity in the hollow cylindrical area 245.

Particle laden dry gas 210 introduces the particles into thedifferential mobility analyzer 200, with a laminar excess gas flow 220introduced so as to minimize turbulent eddies of any sort. An additionaldry gas 230 is introduced at the top of the analyzer 225, ultimatelyexiting the bottom of the analyzer 235 as mobility-classified particleflow 240.

In FIG. 4, only those particles with a particular size 285 are presentlybeing selected. Smaller sized particles 260 impinge on the center tubebefore the detection slit 290. Larger sized particles 280 either impingeafter the slot, or are carried away with the bulk gas flow, but inneither case are measured. Uncharged particles 295, are unaffected bythe electromotive force, thus pass through the exit gas stream 250without passing through the detection slit 290. Mobility spectra maythen be obtained by scanning the high voltage source 205 applied to thecentrally placed tube 270 and cylinder canister 275 through a range ofvoltages corresponding to the sizes of the particles of interest. As thedifferential voltage is scanned each of the three ions will be guided tohit the slit and pass on to a detector located downstream. In thisexample, the small 260, medium 285, and large particles 280 mayrespectively represent HDL, LDL, and VLDL.

The selectable differential mobility analyzer operates by counting thenumber of charged biological particle ions, dn⁺, in a defined samplingtime, dt, at a specific selected size, s, resulting in a size count rateoutput of

${\frac{n^{+}}{t}}_{s}.$

The selectable differential mobility analyzer can be scanned over thedesired specific size range to result in an output of charged biologicalparticle ions counted in the defined sampling time versus size. Sincethe relationship between particle size and density is well understood,the size count rate output above can in turn be converted to data set ofthe number of biological particles counted per second at a specificdensity,

${\frac{n^{+}}{t}}_{\rho},$

versus density, ρ.

We now look at the particular case where the biological particles arelipoproteins. In this case, a scanning size range of 3 nm to 120 nm isappropriate, corresponding to the size of the least and most denselipoproteins present in human patients. Once the scanned data set isproduced in the traditional format of the number of lipoproteinparticles counted per second at a particular size, the specific quantityof lipoprotein versus density can be calculated. By numericallyintegrating the amount of lipoprotein within a density rangecorresponding to the various lipoprotein classes and subclasses,traditional analysis of cardiac heart risk may be assessed.

Data Acquisition and Analysis

The detector 490 used to record the mobility spectrum is a condensationparticle detector, preferably a Model 3025A Condensation ParticleDetector from TSI. The condensation particle detector draws particlessuch as lipoprotein particles through a supersaturated vaporcondensation chamber and allows the vapor to condense onto theparticles, which act as condensation nucleation sites. The device issensitive at the single particle level because every lipoproteinparticle nucleates condensation and gives birth to a solvent droplet. Asingle lipoprotein particle becomes completely covered inside eachcondensation droplet. The condensation process, in effect, magnifies thesize of lipoprotein particles and causes them to grow from 10's of nm indiameter to several μm in diameter.

The μm-scale droplets efficiently scatter light into a photodetector asthey pass through a laser beam. The resultant droplets scatter lightsufficiently to allow for counting individual droplets by measuringchanges in transmission or reflectance (scatter) in the laser beam orother light source. Since only a single lipoprotein particle iscontained in each droplet, this then becomes a method for indirectlycounting single lipoprotein particles.

Lipoprotein particle density measurement in the near-atmosphericgas-phase by particle mobility measurement provides a new way to rapidlydetermine the size distribution of the lipoprotein particles. Referringto FIG. 2, a table is shown including the density and particle size ofthe major lipoprotein subfractions. Since the size and density oflipoprotein particles correlate very well, it is straightforward tocalculate one in terms of the other. This correlation may be shown in aplot of measured diameters and corresponding densities, which are curvefit by a line. This is shown in FIG. 6 and discussed in Section J“Linearity Versus Density.” Thus any lipoprotein particle mobilitymeasurement of a particular size can be related to its correspondingparticle density.

A mobility spectrum of six typical lipoprotein samples obtained fromhuman blood is presented in FIG. 5. Each lipoprotein particle samplesolution, prepared as previously discussed, was diluted to less than10¹¹ lipoprotein particles per mL with a solution of 25 milliMolarammonium acetate. The sample was electrosprayed at a rate of 50 mL permin using a positive 1850 volts to create a well-formed, stableelectrospray. A stable electrospray refers to a lack of visiblefluctuations in the Taylor cone. A flow of 0.5 L per min CO₂ wascombined with 1.0 L per min of dry air to carry the electrospraydroplets through a charge neutralizer, through a differential mobilityanalyzer, and into a condensation particle detector. The resultingspectrum of lipoprotein particles is displayed in FIG. 5, which will bemore thoroughly described below.

Recall that there is a scalar difference between the gel electrophoresislipoprotein sizes and those obtained by differential mobility analysis.The scale factor relates 0.86*gel diameter=ion mobility diameter,shifting the mobility sizes of FIG. 5 down by 86% to correspond with thelipoprotein classes and subclasses of FIG. 2.

Example of a Typical Sample Run

Refer now to FIG. 1 depicting the hardware components involved in thepractice of ion mobility analysis of biological particles, specificallylipoproteins. The sample solution comprises 25 milliMolar ammoniumacetate in an aqueous solution buffered to near neutral pH.

Between 10 and 50 μL of sample solution is introduced into small plasticvial, preferably a 1.5 mL microcentrifuge tube 410. The microcentrifugetube 410 has preferably been ultracentrifuged as previously described inan ultracentrifuge 420, however, other methods such as immunoabsorptionor lectin affinity binding can be used to remove the Lp(a) that wouldotherwise distort the measurement of lipoproteins. The testingmicrocentrifuge tube 450 is in turn installed in a pressurized chamber460 on the electrospray generator. About 3 psig of positive pressure isapplied to the pressurized chamber 460, forcing the sample solution 455to flow through the electrospray capillary 100 and out the beveled tip.At the low differential pressure of 3 psig, it takes several minutes forthe sample to fill the capillary and detect sample 455 exiting thebeveled tip. An improved alternative method to speed up filling thecapillary is to increase the pressurized chamber 460 pressure to about15 psig, which in turn fills the capillary in only about 30 seconds.When the electrospray capillary is filled, the differential pressure isreduced to 3 psig to return the flow rate to about 50 nL per min.

Positive high voltage, from a high voltage variable power supply 430, isthen applied to the sample 455. The high voltage is then scanned from˜5,000 to ˜5,100 volts. The Taylor cone 150 of the capillary 100 is thenexamined during a high voltage 430 scan to find the particular voltageat which the electrospray is stable. A stable electrospray is obtainedwhen the Taylor cone 150 remains fixed in place and points directly awayfrom the axis of the capillary 100, with a sharp tip and a single steadystream of material being ejected. Typically, the microscopic image ofthe Taylor cone appears as a 45° equilateral triangle attached to theflat tip on the end of the tapered capillary 100.

About 1.5 L per minute flow of dry gas 210 is used to introduce thedroplet stream resulting from the Taylor cone 150 into the differentialmobility analyzer 200 through connected tubing, after having been firstcharge reduced with alpha radiation source 480 to either a neutral orsingle positive charge state. 15 L per min of laminar excess gas flow220 is added to the differential mobility analyzer 200. Additional drygas 230 is introduced to collect the mobility selected particles,resulting in a mobility-classified particle flow 240 of 1.5 L per minthat exits the differential mobility analyzer 200, which in turntransfers the mobility selected particles 240 to the condensationparticle counter 490. By scanning the differential mobility analyzer 200high voltage power supply 205, a data set of particles counted persecond versus mobility may be created.

Additional confirmation of a stable electrospray is obtained fromknowledge of the electrospray current. The TSI electrospray aerosolgenerator has a current meter for displaying the electrospray currentoutput by the electrospray high voltage power supply 205. Stableelectrosprays exhibit long term stability with currents varying lessthan ±1 nA from a typical total electrospray current of 300 nA duringthe course of an analysis.

Typically, an essentially pure sample of ammonium acetate buffersolution is run through the instrument to confirm that the capillary isclean. This is called a background spectrum check. Typical backgroundspectra will indicate fewer than 7 particles per mL with particlediameters between 3-4 nm.

The software supplied with the TSI Model 3980 Gas-phaseElectrophoretic-Mobility Macromolecule/nanoparticle Analysis (GEMMA)unit provides several operator defined conditions. The data recordingsoftware allows several choices for the number of size subdivisionsrecorded for each decade of particle diameter. Typically 64 sizesubdivisions per decade are recorded. The scanning range can also beselected. Typically, a scanning range of 3 nm to 100 nm is scanned fortypical LDL samples; however, this range is extended to a higher limitwhen IDL and chylomicrons are measured, to as high as 1200 nm. Scantimes are typically set to 3 min. The position of a peak in an ionmobility spectrum is determined with the manufacturer's software.Computer mouse movement is used to select the maximum value in a peak byobserving a draggable indicator.

Coronary Heart Disease Risk Analysis

Differential mobility spectroscopy of lipoproteins can additionally beused for quick determinations of coronary heart disease (CHD) risk byanalyzing the resultant lipoprotein size distributions.

Plasma was collected from six individuals having lipoprotein patternspreviously characterized by gradient gel electrophoresis. Lipoproteinparticles from these individuals were separated from plasma usingultracentrifugation to isolate components with density less than 1.20g/cm³. The lipoprotein particles were then desalted and analyzed withion mobility spectrometry to determine the biological particle sizedistribution. The results for six types of lipoprotein patterns obtainedby electrospray mobility spectroscopy are shown in FIG. 5.

In FIG. 5, six individual lipoprotein size spectra (510, 520, 530, 540,550, and 560) are presented as the result of differential mobilityanalysis with mobilities converted to size as previously described. Inall but the lowest mass value trace 560, the traces are shiftedvertically in order to differentiate the graphs, which would otherwiseoverlap. To read the vertically shifted mass value, the trace is shiftedvertically to zero mass on its lowest particle diameter. The Massordinate is in relatively scaled arbitrary units of grams.

Pattern A is a designation applied to individuals at relatively low riskfor CHD. Pattern A lipoproteins are characterized by LDL particleslarger (median size of ˜22.5 nm versus ˜20.8 nm) than those obtainedfrom individuals at higher risk for coronary heart disease, i.e.,Pattern B individuals. Additionally, Pattern A lipoproteins arecharacterized by a population of HDL particles that generally showsseveral subtypes. These subtypes are revealed by several peaks in theHDL region (of ˜7-13 nm) for Pattern A spectra. The vertical line atapproximately 8.9 nm indicates the principal HDL peak for patients withPattern A, B, and intermediate Pattern AB. The Pattern a spectra areeasily discerned by observing a second HDL peak at approximately 11 nm.With these characteristics in mind, traces 510, 520 and 530 are low riskType A patterns. The peak in the LDL population of particles for PatternB individuals is smaller by several nanometers than LDL particles fromPattern A individuals. The shift from pattern A to pattern B isrepresented approximately by the vertical lines drawn through the LDLpeaks at approximately 20.8 nm (dashed), and 22.5 nm (solid). The HDLparticles from Pattern B individuals, shown in traces 550 and 560,generally show less heterogeneity; here the HDL peaks for Pattern Bpatterns show only one sharp principal peak, as expected. Thus, the ionmobility measurements agree with gradient gel determinations and providean alternative measurement method for assessing CHD.

Still referring to FIG. 5, trace 540 represents a patient with anintermediate Type AB pattern. This trace 540 shows an LDL peakintermediately disposed between the typical Type A peak at 22.5 nm, andthe typical Type B peak of 20.8 nm. The trace 540 is also distinguishedby only a single HLD peak, typical of the higher risk Type B pattern.

Ion mobility spectroscopy is quantitative and can be used to directlymeasure the total amount of lipoprotein particles in each of thelipoprotein classes and/or subclasses. The area under the curves, in aparticle mass versus independent variable (such as size, density,mobility, etc.) distribution, is directly representative of thelipoprotein particle mass. The measurement technique relies on countingindividual particles as a function of size (diameter). It is thereforepossible the convert the number of particles at a specific size into amass value using the volume and density of the particles. The density oflipoprotein particles is a well-known function of particle size and isobtainable from the literature. The mass values associated with thefigure are simply scaled to indicate relative values but can beconverted to actual mass of lipoproteins in plasma using dilutionfactors along with flow rates of sample and air passing through the ionmobility spectrometer.

The spectra in FIG. 5 also show the existence of lipoprotein particlesthat fall into the VLDL size class. Their presence is indicated by smallbumps in the spectra above particle diameters of about 30 nm. The shadedarea under the plots in this figure are proportional to particle massand this area can be used to assess the mass of particles in any chosenparticle interval such as 30 to 40 nm or 35 to 40 nm or any other choiceof bin size.

Linearity Versus Density

A further example showing how ion mobility analysis can be implementedis shown with the data in FIG. 6. Lipoprotein particles werefractionated with non-equilibrium ultracentrifugation in NaCl/D₂O. Theparticles were removed from the ultracentrifuge tube as liquidfractions, which spanned the range of 1.0 to 1.08 g/cm³. Because theultracentrifugation step was not run until the particles reached anequilibrium flotation position in the ultracentrifuge tube the densityof the lipoprotein particles removed with each fraction is only anestimate, however a reasonable estimate, of the particle's true density,and is displayed as an estimated density. FIG. 6 is a plot oflipoprotein particle diameter as a function of the solution density ofeach fraction reveals the expected inverse linear relationship betweenparticle diameter and density. FIG. 6 has an ordinate of particlediameters in nm as measured by ion mobility analysis, and an abscissa ofestimated particle density in g/cm³ as obtained by non-equilibriumultracentrifugation.

In FIG. 6, the particle diameter was measured using ion mobilityanalysis. The results indicate, that there is an inverse linearcorrelation between particle diameter and particle density, as expected.Data points not exactly matching the inverse linear correlation arethough to be due to the non-equilibrium floatation method used to obtainthe data samples.

Linearity of Lipoprotein Mass Measurements

The linearity of the detection method was verified by analyzing a seriesof dilutions of a specific set of LDL particles. These LDL particleswere isolated by gel filtration from the other classes of lipoproteinparticles. The starting concentration of these LDL particles wasdetermined by measuring the apolipoprotein B concentration of thesolution. A single molecule of apolipoprotein B is present in each LDLparticle. By using the apolipoprotein B concentration it is possible tocalculate the number of LDL particles in the sample. This specificsample was sequentially diluted, and the number of particles in eachdilution (the IMS peak height, #/cm³ axis) was determined bydifferential mobility spectrometry and plotted versus the calculatedapolipoprotein B concentration (the Particles, #/mL axis) at eachdilution. One option available with the commercial software suppliedwith the instrument manufactured by TSI, Inc., plots the number ofparticles detected per cm³ of air passing through the differentialmobility analyzer. Ion mobility peak height is plotted vs. the numberconcentration of LDL particles as determined from apolipoprotein Bmeasurements in FIG. 7. This plot, FIG. 7, shows that the measurement islinear over more than three orders of magnitude from 10¹² to 10¹⁴particles per mL. For increased detail, the inset graph of FIG. 7 showsthe range up to 8·10¹² particles per mL. FIG. 7 implies that variationsin concentrations of lipoprotein subclasses will be accurately measuredover a range of 1000:1, or 0.1%.

Linear Independence of Lipoprotein Analyses

Differential mobility analysis appears to be highly linearly independentover the variety of lipoprotein classes and subclasses. This linearindependence was verified as shown in FIGS. 8-23. In these Figures, asingle human blood sample was fractionated into 16 different averagedensities. The method used was non-equilibrium ultracentrifugation. Onebyproduct of this type of fractionation is that there is always adistribution of densities about an average density. Since we are dealingwith biological particles, there are wide ranges of sizes comprising inthe fractions having a particular average densities. An attempt was madeto select density fractions representing each of the lipoproteinparticle subclasses.

FIGS. 8-23 show resultant differential mobility analysis scans of 16fractions of human lipoprotein over a range of selected averagedensities spanning the various human lipoprotein classes and subclasses.Lipoprotein particles were prepared as above from human plasma, thensubjected to ultracentrifugation. A density gradient in anultracentrifuge tube was prepared with NaCl dissolved in D₂O. The choiceof gradient composition provided a way to separate lipoprotein particleswith average densities varying between about 1.000 (FIG. 8) and 1.090g/cm³ (FIG. 23) from other material in the plasma. For referencepurposes, gel electrophoresis gradient 8 is used to separate averagedensities over a range from about 1.000 (FIG. 8) to 1.062 g/cm³(bracketed between FIGS. 19 and 20). This density gradient distributeslipoprotein particles along the length of the ultracentrifuge tube.Lipoproteins with higher density such as HDL move to the bottom of theultracentrifuge tube, as do protein molecules. Lower densitylipoproteins such as VLDL rise to the top of the tube during theultracentrifugation process.

The resulting liquid in the ultracentrifuge tube was distributed into 16fractions, which were subsequently dialyzed against 25 mM ammoniumacetate for 3 hours using a 10,000 molecular weight cut-off dialysismembrane.

The size distribution of the lipoprotein particles in each fraction wasthen determined using ion mobility spectral analysis. These sizedistributions appear in FIGS. 8-23, and have densities that correspondto lipoprotein classes and subclasses as indicated in VLDL I (FIGS.8-10) and II (FIG. 11), IDL I (FIG. 12) and II (FIG. 13), and LDL I(FIG. 14), IIa (FIG. 15), IIb (FIG. 16), IIIa (FIG. 17), IVa (FIGS. 18and 19), and HDL IIa (FIGS. 20-23). The fractions of FIGS. 8-23, andtheir subsequent density analyses, are somewhat confusing since theoriginal laboratory sample labels of fractions are respectively 1 a, 1b, 2 a, 2 b, 3-10, 11 a, 11 b, 12 a, and 12 b. Consequently, the samplelabels bear some resemblance to the lipoprotein subclasses. Lipoproteinsubclass 111 b was not shown in this selection of fractions, althoughbased on the complement of FIGS. 8-23, the IIIb distribution should besimilarly peaked within a range of 1.041 and 1.044 g/cm³ as indicated onFIG. 2. Each particle size distribution is plotted as mass vs. particlediameter. In each Figure, the mass value refers to the mass of particlesper m³ at each indicated particle diameter where m³ is referenced to thevolume of lipoprotein-laden gas entering the differential mobilityanalyzer. Although only single positively charged particles are measureddue to the selective electromotive classification used by the DMA, theμg/m³ number is corrected for the fraction of particles that are chargedin any given particle size interval. This rate does not take intoaccount either the rate at which a sample solution is electrosprayed, orthe flow rate of flow gas that sweeps the particles towards the DMA.This mass value can be converted into the mass of lipoprotein particlespresent in the original sample plasma by using the information of thegas flow rate, flow rate of electrosprayed sample, particle transferefficiency and liquid dilution factors.

FIGS. 9-18, 20, 21, and 23 indicate that for lipoprotein densitiesranging from about 1.003-1.090 g/cm³, in this particular human sample,each density sample through ultracentrifugation results in one principalpeak in the mass versus size graph.

For FIGS. 8, 19, and 22 there is some bimodality and broadening of thepeaks. It is not presently understood what these broadened and bimodallipoprotein distributions represent. It is possible that this bimodalitycould be an artifact of the sample preparation method discusses above.That is, the sample preparation technique could be disruptive enough tobreak apart some of the agglomerated biological particles, resulting insmaller sized fragments. If this is the case, then a modification of thesample preparation method could then resolve these samples intorelatively clean single peak size spectra as well.

The bimodality and broadening could also be an artifact resulting fromthe nonequilibrium ultracentrifugation used to generate the subfractionsin this particular experiment. This issue will be resolved by furtherexperiment.

Nonequilibrium ultracentrifugation also results in the non-steady statedensities determined in FIGS. 8-23. As such, all densities in theseFIGS. 8-23 are approximate.

Linear Superposition of Lipoprotein Subclasses

In principal, for lipoproteins represented in these subclasses, anentire plasma sample could be mathematically represented by the linearsuperposition of each lipoprotein subclass. Even if, ultimately, themultiple peak phenomenon remains, then traditional numerical methods ofdata correlation such as least squares or singular value decompositioncould be used to determine the amount of each lipoprotein subclass in anentire sample. Thus, in a single differential mobility spectral scan,the quantity of each lipoprotein subclass a plasma sample could bedetermined. After making this determination, both the quantity and typeof each lipoprotein would be known. By statistically correlating theresulting subclass information with population mortality and riskfactors, a more accurate assessment of coronary heart disease risk wouldresult. In particular, the known characteristics of a bimodal vs.unimodal HDL distribution, and the peak lipoprotein diameter of the LDLdistribution, could be readily transformed into a risk factor rangingfrom 100% Type A pattern (low risk) to 100% Type B pattern (high risk).

CONCLUSION

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application were eachspecifically and individually indicated to be incorporated by reference.

The description given here, and best modes of operation of theinvention, are not intended to limit the scope of the invention. Manymodifications, alternative constructions, and equivalents may beemployed without departing from the scope and spirit of the invention.

1. A method for assessing a lipid-related health risk in a patient, themethod comprising: (a) isolating lipoprotein particles from a biologicalsample taken from a patient; (b) delivering the lipoprotein particles ina charged state to a differential mobility analyzer; (c) obtaining amobility distribution of the charged lipoprotein particles; (d)comparing the mobility distribution of the charged lipoprotein particlesfrom the patient to a reference lipoprotein particle mobilitydistribution; and (e) determining whether the patient has thelipid-related health risk based on the comparison.
 2. The method ofclaim 1, wherein the lipoprotein particles are put into a charged stateby electrospraying the lipoprotein particles.
 3. The method of claim 1,wherein the lipoprotein particles comprise at least one lipoproteinselected from the group consisting of VLDL, IDL, LDL, HDL, and theirsubclasses.
 4. The method of claim 1, wherein the biological sample isblood, plasma or serum.
 5. The method of claim 1, wherein thelipoprotein particles from the patient are classified on the basis ofthe mobility distribution.
 6. The method of claim 1, further comprisingconverting the mobility distribution of lipoprotein particles to atleast one measurement selected from the group consisting of a particlemobility, a particle size, a particle density, a particle mass, a numberof particles in a size interval, and an amount of particle mass in asize interval, prior to determining the lipid-related health risk of thepatient.
 7. The method of claim 1, wherein the lipid-related health riskis hyperlipidemia.
 8. The method of claim 7, wherein the hyperlipidemiais hypertriglyceremia.
 9. The method of claim 1, wherein thelipid-related health risk is the risk for a cardiovascular condition ordisease, or the risk for a predisposition to develop a cardiovascularcondition or disease.
 10. The method of claim 9, wherein thecardiovascular disease is coronary heart disease.
 11. A method fordetermining a lipid-related health risk in a patient, the methodcomprising: (a) obtaining lipoprotein particles from a patient sample,wherein the patient is suspected of having a lipid-related health risk;(b) delivering lipoprotein particles in a charged state to adifferential mobility analyzer; (c) obtaining a mobility distribution ofthe charged lipoprotein particles; (d) comparing the mobilitydistribution of the charged lipoprotein particles from the patient to areference lipoprotein particle mobility distribution taken from apatient that does not have the lipid-related health risk; and (e)determining whether the patient has the lipid-related health risk basedon the comparison.
 12. The method of claim 11, wherein the chargedlipoprotein particles comprise at least one lipoprotein selected fromthe group consisting of VLDL, IDL, LDL, HDL, and their subclasses. 13.The method of claim 11, wherein the patient sample is blood, plasma orserum.
 14. The method of claim 11, wherein the charged lipoproteinparticles from the patient are classified on the basis of the mobilitydistribution.
 15. The method of claim 11, further comprising convertingthe mobility distribution of lipoprotein particles to at least onemeasurement selected from the group consisting of a particle mobility, aparticle size, a particle density, a particle mass, a number ofparticles in a size interval, and an amount of particle mass in a sizeinterval, prior to determining the lipid related health risk of thepatient.
 16. The method of claim 11, wherein the lipid-related healthrisk is hyperlipidemia.
 17. The method of claim 16, wherein thehyperlipidemia is hypertriglyceremia
 18. The method of claim 11, whereinthe lipid-related health risk is the risk for a cardiovascular conditionor disease, or the risk for a predisposition to develop a cardiovascularcondition or disease.
 19. The method of claim 18, wherein thecardiovascular disease is coronary heart disease.
 20. The method ofclaim 11, wherein the lipoprotein particles are put into a charged stateby electrospraying the lipoprotein particles.